The long-term goal of this study is to test an oral vaccine(s) for strain 0139 cholera organisms. The immediate goal is to validate the use of a standardized frozen inoculum when challenging volunteers with virulent cholera bacteria. This will allow the development of a multi-center vaccine study. Healthy volunteers were recruited for an inpatient, dose-escalation challenge with live virulent cholera organisms (V. cholerae, 0139, strain 4260B). The study goal was to identify a dose at which the attack rate was greater than or equal to 80%, and the geometric mean purge rate exceeded 3L during the defined study period. We have completed five inpatient studies. In the first two studies, the volunteers were challenged with 105 organisms of frozen, reconstituted, virulent V. cholerae, 0139, strain 4260B. Although the attack rate was 8/10, the geometric mean purge was insufficient (2175 g). Therefore, we escalated the dose for subsequent challenges to achieve 1 x 106 organisms, as described in the protocol. At this higher dose, the disease severity was adequate to establish a challenge model. We demonstrated an attack rate of 14/15 at a dose of .1 x 106 CFU. The geometric mean purge of 5621 mL was also adequate by protocol criteria (> 3L). Thus, we have met the study criteria to complete the protocol at CHMC with this dose. Confirmation of these results is planned for a subsequent study at the Center for Vaccine Development, University of Maryland.